TRIAMPO ET AL. 1

Effects of Static Magnetic Field on Growth of Leptospire, Leptospira

interrogans serovar canicola: Immunoreactivity and Cell Division

WANNAPONG TRIAMPO,1,4* GALAYANEE DOUNGCHAWEE,2 DARAPOND

TRIAMPO, 3 JIRASAK WONG-EKKABUT, 1 and I-MING TANG1,4

Department of Physics, Faculty of Science, Mahidol University, Bangkok 10400,

Thailand,1Department of Pathobiology, Faculty of Science, Mahidol University,

Bangkok 10400, Thailand,2Department of Chemistry, Faculty of Science, Mahidol

University, Bangkok 10400, Thailand,3Capability Building Unit in Nanoscience and

Nanotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand4

*Corresponding author. e-mail: scwtr@mahidol.ac.th ; wtriampo@yahoo.com

phone:+662-889-2337 fax: +662-354-7159

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Abstract: The effects of the exposure of the bacterium, Leptospira interrogans

serovar canicola to a constant magnetic field with magnetic flux density from a

permanent ferrite magnet = 140±5 mT were stud ied. Changes in Leptospira cells after

their exposure to the field were determined on the basis of changes in their growth

behavior and agglutination immunoreactivity with a homologous antiserum using dark-

field microscopy together with visual imaging. The data showed that the exposed

Leptospira cells have lower densities and lower agglutination immunoreactivity than

the unexposed control group. Interestingly, some of the exposed Leptospira cells

showed abnormal morphologies such as large lengths. We discussed some of the

possible reasons for these observations.

Key words : Leptospirosis, Leptospira interrogans, magnetic field, dark-field

microscopy, immunoreactivity, cell division

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INTRODUCTION

Leptospirosis is an acute febrile illness caused by pathogenic spirochete bacteria

of the genus Leptospira (1, 2). This disease has emerged as an important public health

problem worldwide. The symptoms of this disease can range from mild-flu- like

symptoms to severe (often fatal) complications such as renal and/or liver failure and

hemorrhage (referred to as Weil’s syndrome) (3). Most outbreaks tend to be seasonal

in nature and are often associated with environmental factors, animals, and agricultural

and occupational cycles such as rice cultivation in marshy lands. Mammals such as rats

and cattle are commonly involved in the transmission of this disease to humans via

direct or indirect exposure to contaminated tissues or urine (1, 2, 4). Out-breaks of

leptospirosis occur mainly after flood, making it an occupational hazard for sanitary

and agricultural workers, as well as a recreational hazard for humans (5). Some

pathogenic leptospira species have also been found to be associated with domesticated

animals. For example, serovar canicola (Leptospira canicola) has adapted itself to

canines; therefore, it has become common in many human communities. Although

there has been no report of leptospirosis in canines in Thailand, there is a great potential

for the transmission of the disease between humans and dogs kept as household pets,

unless one is aware of the disease.

L. canicola cells used in our study are motile aerobes that are very thin, flexible

and spiral-shaped of about 0.1 µm width and 6-20 µm length. Leptospira cells are

difficult to observe under a light microscope. They can, however, be observed by dark-

field microscopy using wet samples. This allows for the determination of agglutination

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immunoreactivity to be determined. The leptospiral outer membrane or surface

antigens can be detected through its agglutination with a homologous [antiserum]. The

optimal conditions for its growth and as well, its biology are well documented in the

literature (1, 2). Moist environments with a neutral pH are suitable conditions for the

survival of leptospira outside the host. The optimal cultivation temperature is

approximately 20-32°C. In general, Leptospira species are highly susceptible to

adverse environmental conditions such as exposure to dry air, chemicals such as

chlorine or iodine in detergents, unfavorable pH ( > 8.0 or < 6.5), strong

electromagnetic fields and high temperatures (above 40°C).

Magnetic fields (MFs) also affect various biological functions of living

organisms, for example, DNA synthesis and transcription (6), as well as ion

transportation through cell membranes (7). Almost all living organisms are exposed to

magnetic fields from various sources. The geomagnetic field on the surface of the earth

is approximately 0.50-0.75 gauss in strength. There have been several studies on the

effects of exposure to MFs and several of these have given rise to controversies over

the past decades. The growth rate of the Burgundy wine yeast has been shown to

decrease when an extremely low magnetic flux density (MFD) of 4 gauss is applied (8).

The growth of Trichomonas vaginalis is accelerated when it is exposed to 460-1200

gauss (9). The growth rate of Bacillus subtilis increases when exposed to 150 gauss

and decreases when exposed to more than 300 gauss (10). Similar results were reported

for Chlorella; an exposure of less than 400 gauss increases the growth, while exposure

to 580 gauss decreases the growth rate (11). Several studies point to the MF as a factor

influencing the growth and survival of living organisms, which vary at different MFDs

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(12, 13, 14, 15). Other researchers have studied the effects of MFs on bacteria at the

enzyme (16) or genetic (17) level.

To study the efficacy of using magnetic field to control or prevent the growth of

Leptospira, we applied MF on selected Leptospira cells at various intensities and

exposure duration levels. We then determined the agglutinating activity of

experimental bacteria using dark field microscopy.

MATERIALS AND METHODS

Pathogenic Leptospira interrogans, serovar canicola was used in this study.

Bacteria l cells were grown in the Ellinghausen and McCullough modified by Johnson

and Harris [EMJH] liquid medium (2). The bacterial cells were grown at a temperature

of 27±1°C in the dark.

A cylindrical permanent ferrite magnet 5 cm in diameter was placed beside 15

ml culture glass tube (less than 1 ml apart) containing 1 ml of a suspension of newly

subcultured leptospira cells in the EMJH liquid medium. MF and homogeneity of

5140 ± mT (northpole) were checked using a teslameter (Hall effect Teslameter digital,

order no. 13610.93; Phywe Systeme Gottingen, Germany). The intensity of static

magnetic field used in our experiments was chosen on the basis of Genkov et al. (9)

findings. Genkov et al. had used more or less this intensity of a constant MF to induce

the growth and development of Trichomanas vaginalis. For this type of exposure, no

shielding against the natural variations of terrestrial MF was required, the value of

approximately 0.050 mT is negligible with respect to the MF intensities applied. An

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experiment using cells not exposed to MF was simultaneously performed as the control,

which was placed at a distance of about 100 cm from the exposed group.

In the absence of magnets, MFD was 0.05±0.01 mT. All bacterial samples

were exposed to MF for different durations, that is, 0 (control sample), 1, 2, 3, 4, 5, and

6 d. After MF exposure, individual samples were further incubated for 7 d.

Immediately after 7 days of incubation, dark-field micrographs were taken using a CCD

camera to observe cell development. The growth and agglutination properties using the

microscopy agglutination test (MAT) with a homologous antiserum and

immunoreactivity were scored as follows:

4+ = 100 % absence of Leptospira cells from the field

3+ = 75 % absence of Leptospira cells from the field

2+ = 50% absence of Leptospira cells from the field

1+ = 25% absence of Leptospira cells from the field

stet

MAT has been commonly used as a diagnostic tool for leptospirosis. This may not be

the most reliable test. It, however, is arguably the most appropriate test for this study.

The same set of conditions and specimens were used in the experiments, which were

repeated twice.

Atomic Force Microscopy (AFM) and sample preparation Scanning probe

microscopy (SPM) (Digital Instruments Veeco Metrology Group, New York, USA)

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was used for AFM surface morphology imaging. Images were acquired in the contact

mode showing height contours that highlight the spiral shape and fine surface

morphology of Leptospira cells. An AFM scanner with hardware correction for the

nonlinearities of the piezoelectric element was used. The scanner has a maximum xy

range of 125 by 125 µm and a z range of 6µm. The cantilevers of Si3N4, 125 µm long

and 35 µm wide with a spring constant of 0.58 Nm-1 were used. To locate the area of

interest in the samples and identify any bacteria, we used a built- in long-range on-axis

microscope, capable of a 5:1 zoom and x 3,500 magnification. Imaging was carried out

at scan speeds between 1 and 50 µm/s. Images were acquired at 256x256 pixels. A

typical imaging session began using a built- in optical microscope and by moving the x-

y table to search for bacterial cells. The AFM cantilever was then moved forward to the

surface close to the chosen bacterial cell.

Each sample was prepared using the method described above. It was then

dropped on a microscope glass slide and dried in air.

RESULTS

Figure 1 shows the AFM picture of a L. interrogans serovar canicola cell taken

with a Digital Instrument Nanoscope IIIa (Digital Instruments Veeco Metrology Group,

New York, USA) in the contact mode. The image shows a normal morphology of L.

interrogans serovar canicola, that is, the spiral shape. It is worth noting that AFM

usually reveals the actual roughness of the surface of the bacteria l envelope. Other

types of microscopy frequently show the surface to be relatively smooth. This

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technique was also used to observe the surface morphology of bacterial cells before and

after the exposure to MF. It should be noted that this image does not demonstrate the

rough envelope very clearly. However, it does show the normal bacterial morphology.

Figure 1

Figure 2 shows some representative dark field micrographs of L. interrogans

serovar canicola taken at the logarithmic growth phase (at 1:10 dilution of culture

samples) and for different durations of MF exposure, that is, 0, 2, 3, and 6 d. After 7 d

of incubation, the samples were observed under a dark field microscope and images

were taken using a CCD camera. Even though there are some noises in the images, the

inhibition of cell growth could be observed. The implications of these observations are

significant given the results of other studies(6-17). From Figs. 2A to 2D, one can

clearly observe that cell density decreased with exposure time, particularly after more

than 3 d. This indicates the decrease in growth rate resulting in the decrease in the

number of bacterial cells. This is one of the factors that explain the lower agglutination

immunoreactivity, which is there were fewer remaining living bacteria l cells to

agglutinate.

Figure 2

Figure 3 shows the dark field micrographs of agglutinated bacteria l cells after

reacting with the specific antiserum; Fig.3A shows a complete agglutination (100%

immuno) and Fig.3B shows 50% agglutination (with only one half of free- living

bacterial cells present).

Figure 3

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On the basis of the criteria mentioned at the end of the previous section, the

agglutination reactivities of the L. interrogans serovar canicola exposed to different

intensities of MF are listed in Table 1 (with longer exposure time, the leptospiral

bacterial cells demonstrated a lower agglutination immunoreactivity than that of the

reference antiserum tested. The end point of reactivity was 50% agglutination (2+)).

The agglutination immunoreactivity score decreased with exposure time of Lectospire

cells as shown in Fig 4. Comparing the MAT results of control Leptospire cells (0 d

exposure) and those of bacteria l cells after exposed to MF, we found that the latter

groups (particularly those with longer exposure) showed lower agglutination reactivies.

These findings may indicate the presence of a lower amount of agglutinin or number

(density) of Leptospires cells in the exposed samples than in the control samples. It

should be emphasized that the same set of conditions and specimens were used in the

experiments that were repeated twice, and the experiments yields exactly the same

(semiquantitative) results. The scoring data therefore did not show an error. Once again,

each experimental setup, it has one control (nonexposed) group and six exposed groups

with different durations of exposure.

Table1

Figure 4

Besides the decrease in the number of Leptospira cells as the cause of the

decrease in agglutination immunoreactivity as mentioned above, the “denaturing effect”

of the antigen-antibody reaction may be an other contributing factor to this

phenomenon, which can be explained as follows: Typically, antibodies are large soluble

TRIAMPO ET AL. 10

protein molecules known as immunoglobins and are produced by B-cells. They bind to

specific antigens in a lock-and-key fashion (lock = antibody; key = antigen) (18). Their

shape should, therefore, be specific to particular antigens. When a specific antibody

encounters an antigen, it will form an antigen-antibody complex through some

noncovalent forces such as electrostatic force, hydrogen bond, van der Waal force or

hydrophobic force. When a change in what of a single atom occurs, the complex can

become unbound. This specificity could be the underlying factor for the denaturation

of the antigen-antibody reaction. Under the conditions used in the study, the motion or

transfer of any electrons or ions onto the cell membrane could induce an electric

current. This current may perturb the other charge particle motion in the cell thus

resulting in the loss of binding (19).

Figure 5

Surprisingly, we observed that some Leptospira cells exposed for 3 or more

days were longer than the control bacterial cells (see Fig. 5). This preliminary finding

probably indicates that there is some disturbance in cell division. More experiments

must be carried out to examine and determine the exact mechanism underlying these

observed phenomena. Our present explanation for this abnormality in cell division is

based on the following: Like most bacteria and archaea, Leptospira cells divide

symmetrically possibly via the formation of a septum in the middle of the cell (we

consider that binary fission is less likely). For the time being, we use AFM in the

investigation of division-related morphologies. Recent evidence indicates that

synthesized proteins dedicated to cell division are assembled between segregated

chromosomes at an appropriate time (20). The key to this assembly is the filamentous

TRIAMPO ET AL. 11

temperature exposure sensitive (Ftsz structural) analogue of tubulin (21). DNA damage

caused by MF exposure induces mutation, resulting in the abnormal synthesis of FtsZ,

which in turn could interfere or stop cell division. Similar to previous studies of

Escherichia coli, FtsZ appears to induce the earliest (known) step in cell division.

E.coli cells with a mutation of ftsz caused by exposure to certain conditions do not

divide. This result in the formation of long filamentous cells that can replicate and

segregate their chromosomes (22).

Our finding is at least the first step toward a grater understanding of this the

development of diagnostics, treatment, and prevention schemes for bacterium and

leptospirosis. We hope that further studies of leptospirosis will lead to this disease in

the near future.

ACKNOWLEDGMENTS

This research was supported in part by the Thailand Research Fund,

TRG4580090 and RTA4580005 and MTEC Young Research Group funding MT-NS-

45-POL-14-06-G. The support of the Royal Golden Jubilee Ph.D. Program

(PHD/0240/2545) to Jirasak Wong-ekkabut and I-Ming Tang is also acknowledged.

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FIG. 1. Atomic force micrograph (AFM) of Leptospira interogans serovar canicolataken using Digital Instrument NanoScope IIIa in the contact mode under control conditions, that is, without MF exposure. Scan size was 20 µm and scan rate was 1 Hz. It shows a spiral-shaped leptospire of approximately 10 - 20µm.

TRIAMPO ET AL. 16

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FIG. 2. Dark field micrographs of Leptospira interrogans serovar canicola exposed toMF for different durations. The images were taken at the log phase of each experimental culture sample (diluted 1:10 of original).

TRIAMPO ET AL. 18

A B

FIG. 3. Dark field micrographs of agglutinated bacterial cells after reacting

with homologous antiserum, showing complete agglutination (100% reactivity ;

A) and 50% agglutination with one-half of free- living bacteria l cells remaining

(B).

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TABLE 1. Agglutination characteristics of leptospires after magnetic field exposure

for various durations.

Exposureduration (d)

1:50dilution

1:100dilution

1:200dilution

1:400dilution

1:800dilution

1:1600dilution

1:3200dilution

0a 4+ 3+ 2+ 2+ 2+ 2+ 1+1 3+ 2+ 1+ - - - -2 3+ 2+ 1+ - - - -3 2+ - - - - - -4 2+ - - - - - -5 1+ - - - - - -6 NA - - - - - -

a Representive sample of control unexposed leptospires showing a higher MAT titer

(1:1600) than exposed samples for various durations.

NA indicates no agglutination occurred.

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Agglutination immunoreactivity

0

1

2

3

4

5

0 1 2 3 4 5 6 7

Exposure duration (d)

Agg

luti

atin

g ac

tivi

ty

FIG. 4. Plots of data shown in Table 1.

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FIG. 5. Dark-field micrographs of L. interrogans serovar canicola taken at the

same magnification (x200). Control sample unexposed to magnetic field; the

leptospires have an approximate length of 10-20 µm (A) compared with

magnetic field-exposed leptospires (B) with some cells longer than others.

Circles indicate individual bacterial cells.